Background Selenocysteine tRNAs (tRNASec) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNASec- interacting factors are incompletely understood. Methodology/Principal Findings We applied rational mutagenesis to obtain well diffracting crystals of murine tRNASec. tRNASec lacking the single-stranded 3′-acceptor end (ΔGCCARNASec) yielded a crystal structure at 2.0 Å resolution. The global structure of ΔGCCARNASec resembles the structure of human tRNASec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNASec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2′-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNASec in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNASec. Conclusions/Significance We provide the most highly resolved structure of a tRNASec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNASec support its interaction with proteins.
